Polymethine compounds with long stokes shifts and their use as fluorescent labels

ABSTRACT

The present disclosure relates to new compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications. The labels are advantageous due to their long Stokes shifts.

The present disclosure relates to new polymethine compounds and theiruse as fluorescent markers. In particular the compounds may be used asfluorescent labels for nucleotides in nucleic acid sequencingapplications.

BACKGROUND

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this disclosure pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Non-radioactive detection of nucleic acids utilizing fluorescent labelsis an important technology in molecular biology. Many proceduresemployed in recombinant DNA technology previously relied heavily on theuse of nucleotides or polynucleotides radioactively labelled with, forexample ³²P. Radioactive compounds permit sensitive detection of nucleicacids and other molecules of interest. However, there are seriouslimitations in the use of radioactive isotopes such as their expense,limited shelf life and more importantly safety considerations.Eliminating the need for radioactive labels enhances safety whilstreducing the environmental impact and costs associated with, forexample, reagent disposal. Methods amenable to non-radioactivefluorescent detection include by way of non-limiting example, automatedDNA sequencing, hybridization methods, real-time detection ofpolymerase-chain-reaction products and immunoassays.

For many applications it is desirable to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes. In suchmultiplex methods the number of reaction vessels may be reduced,simplifying experimental protocols and facilitating the production ofapplication-specific reagent kits. In multi-colour automated DNAsequencing for example, multiplex fluorescent detection allows for theanalysis of multiple nucleotide bases in a single electrophoresis lane,thereby increasing throughput over single-colour methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

However, multiplex fluorescent detection can be problematic and thereare a number of important factors which constrain selection offluorescent labels. First, it may be difficult to find dye compoundswhose emission spectra are suitably spectrally resolved in a givenapplication. In addition when several fluorescent dyes are usedtogether, to generate fluorescence signals in distinguishable spectralregions by simultaneous excitation may be difficult because theabsorption bands of the dyes which could be useable for this are usuallywidely separated, so it is difficult to achieve more or less equalfluorescence excitation efficiency even for two dyes. Many excitationmethods use high power light sources like lasers and therefore the dyemust have sufficient photo-stability to withstand such excitation. Afinal consideration of particular importance in molecular biologymethods is the extent to which the fluorescent dyes must be compatiblewith the reagent chemistries used such as for example DNA synthesissolvents and reagents, buffers, polymerase enzymes and ligase enzymes.

As sequencing technology advances a need has developed for furtherfluorescent dye compounds, their nucleic acid conjugates and dye setswhich satisfy all of the above constraints and which are amenableparticularly to high throughput molecular methods such as solid phasesequencing and the like.

Fluorescent dye molecules with improved fluorescence properties such asfluorescence intensity, shape and wavelength maximum of fluorescenceband can improve the speed and accuracy of nucleic acid sequencing.Strong fluorescence signal is especially important when measurements aremade in water-based biological buffers and at higher temperature as thefluorescence intensity of most dyes is significantly lower at suchconditions. Moreover, the nature of the base to which a dye is attachedalso affects the fluorescence maximum, fluorescence intensity and othersspectral dye properties. The sequence specific interactions between thenucleobases and the fluorescent dyes can be tailored by specific designof the fluorescent dyes. Optimisation of the structure of thefluorescent dyes can improve the efficiency of nucleotide incorporation,reduce the level of sequencing errors and decrease the usage of reagentsin, and therefore the costs of, nucleic acid sequencing.

Improvements in the detection in particular of multiple fluorescentlabels can be achieved using fluorescent dyes with different and,especially, with bigger than ordinary Stokes shifts.

The Stokes shift is the difference between the absorption maximumwavelength and the emission maximum wavelength for the same electrontransition.

Most fluorescent dyes in the visible region have a Stokes shift lessthan 40 nm, meaning the most efficient excitation wavelength and maximumof emission wavelength are relatively close together. Compounds withlonger Stokes shift have better signal to noise ratio as the emissionand excitation wavelength are further apart. Long Stokes shift dyes alsoallow two different labels to be separately detected using the sameemission channel but with different excitation wavelengths. For examplea detection measurement can be recorded between say 550-570 nm, and candetect signals arising from a first label with a short Stokes shiftwhich is excited at 532 nm, and a second label which has a long Stokesshift and is excited at say 450 nm

Described herein are improved polymethine constructs having long stokesshifts, and their use as bio-molecule labels, particularly as labels fornucleotides used in nucleic acid sequencing. Particular improvements canbe seen in the efficiency of labelled nucleotide incorporation andlength of sequencing read obtainable using the new fluorescentconstructs when detecting measurements in a single detection channel.

SUMMARY

According to a first aspect this disclosure provides compounds of theformula (I) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, hydroxy, alkoxy, amino or afurther ring fused to an adjacent carbon atom where the ring may containSO₃ ⁻, sulfonamide, halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, hydroxy, alkoxy, amino, COOH or anamide or ester thereof and n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl,or substituted aryl; andRe₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rc₁, Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or esterthereof.

This disclosure provides compounds of the formula (I′) or mesomericforms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, hydroxy, alkoxy, amino or afurther ring fused to an adjacent carbon atom where the ring may containSO₃ ⁻, sulfonamide, halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, hydroxy, alkoxy, amino, COOH or anamide or ester thereof and n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl,or substituted aryl; andRe₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rc₁, Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or esterthereof.

In certain examples where Re₁ is alkyl, Rb₁, Rc₁ can be COOH or COO⁻ oran amide or ester thereof.

In certain examples n can be 0 such that the phenyl group isunsubstituted, and Re₁ contains a COOH or COO⁻ or an amide or esterthereof.

In another embodiment the compounds of the present disclosure can beconjugated with a variety of substrate moieties such as, for example,nucleosides, nucleotides, polynucleotides, polypeptides, carbohydrates,ligands, particles, cells, semi-solid surfaces (e.g. gels) and solidsurfaces. The conjugation can be carried out via the carboxy or sulfonicgroup on Rc₁, Re₁ or Rb₁ which can be turned into an amide, sulfonamideor ester.

According to a further aspect of the disclosure therefore, there areprovided dye compounds comprising linker groups to enable, for example,covalent attachment to such substrate moieties such as nucleotides.

According to a further aspect the disclosure provides a nucleoside,nucleotide or oligonucleotide compound defined by the formula: N-L-Dye,wherein N is a nucleotide, L is an optional linker moiety and Dye is afluorescent compound according to the present disclosure.

The nucleotide may be a nucleotide 5-triphosphate.

In a further aspect the disclosure provides methods of sequencing usingthe dye compounds of the present disclosure.

According to a further aspect the disclosure also provides kitscomprising dye compounds (free or in conjugate form) which may be usedin various immunological assays, oligonucleotide and nucleic acidlabelling and for DNA sequencing by synthesis. In yet another aspect thedisclosure provides kits comprising dye ‘sets’ particularly suited tocycles of sequencing by synthesis on an automated instrument platform.

A further aspect of the disclosure is the chemical preparation ofcompounds of the disclosure.

DETAILED DESCRIPTION

This disclosure provides novel compounds particularly suitable formethods of fluorescence detection and sequencing by synthesis. Novelcompounds having an N-phenyl indole moiety are advantageous influorescence maximum position, their intensity and photostabilitycompared to N-alkyl analogues and therefore improve certain nucleic acidsequencing applications.

According to a first aspect the disclosure provides compounds of theformula (I) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or a further ring fused to anadjacent carbon atom where the ring may contain SO₃ ⁻, sulfonamide,halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl; andRe₁ is alkyl, substituted alkyl, aryl or substituted aryl;wherein either Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or esterthereof.

This disclosure provides compounds of the formula (I) or mesomeric formsthereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, hydroxy, alkoxy, amino or afurther ring fused to an adjacent carbon atom where the ring may containSO₃ ⁻, sulfonamide, halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, hydroxy, alkoxy, amino, COOH or anamide or ester thereof and n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl,or substituted aryl; andRe₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rc₁, Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or esterthereof.

The length of chain x may be 0, 1 or 2. The chain may have one carbonand one double bond, three carbons and two double bonds or five carbonsand three double bonds. X may be 1 such that the chain contains three CHgroups.

The molecules may contain a sulphonamide or SO₃ ⁻ moiety at position Ra.Ra₁ may be sulphonamide. The sulphonamide can be SO₂NH₂ or SO₂NHR, whereR is an alkyl, substituted alkyl, aryl or substituted aryl group. Ra₁can be H. Ra₁ can be SO₃ ⁻. Ra₁ can be a further ring fused to anadjacent carbon atom. The Ring may be substituted or unsubstituted. Thering may be substituted with one or more sulphonamide or SO₃ ⁻ groups.The sulphonamide can be SO₂NH₂ or SO₂NHR, where R is an alkyl,substituted alkyl, aryl or substituted aryl group.

Ra₁ can be a further aliphatic, aromatic or heterocyclic ring fused toadjacent carbons of the indole ring. For example, in such cases when anaromatic ring is fused the dyes end group can represent a structure oftype

where Rf can be H, alkyl, substituted alkyl, aryl, substituted aryl,halogen, carboxy, sulphonamide, or sulfonic acid. In such structures Rfcan appear multiple times, for example Rf can be both H and SO₃ ⁻, orcan be multiple SO₃ ⁻ groups.

Thus the dyes of the disclosure can be described by Formula (1A) or(1A′):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl;Re₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or ester thereof;andwhere Rf can be one or more of H, alkyl, substituted alkyl, aryl,substituted aryl, halogen, carboxy, sulphonamide, or sulfonic acid.

Thus the dyes of the disclosure can be described by Formula (IB) or(IB′):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl;Re₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or ester thereof.

Thus the dyes of the disclosure can be described by Formula (IC) or(IC′):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl;Re₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or ester thereof.

Thus the dyes of the disclosure can be described by Formula (ID) or(ID′):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;q is 1-6;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3;Rc₂ is alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl;Re₁ is alkyl, substituted alkyl, aryl or substituted aryl; whereineither Rb₁ or Re₁ contains a COOH or COO⁻ or an amide or ester thereof.

Thus the dyes of the disclosure can be described by Formula (IE) or(IE′):

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion andm is an integer 0-3;x is an integer 0-2;q is 1-6;Rb₁ is SO₃ ⁻, sulphonamide or halogen, and n is 0-3;Rc₂ is alkyl or substituted alkyl;each of Rd₁ and Rd₂ is independently H, alkyl or substituted alkyl;Re₁ is alkyl, substituted alkyl, aryl or substituted aryl; andX is OH, O⁻ or an ester or amide.

The carboxy group or its derivatives is attached either position Rb₁ Rc₁or Re₁. When attached to Rb₁, Re₁ can be unsubstituted alkyl, or alkylwith one or more substituents. The COOH group can act as a linkingmoiety for further attachment or is linked to a further molecule. Onceconjugation has occurred, the COOH or COO is turned into an amide orester.

Alternatively, Rc₁ can contain a carboxy group. The carboxy group can beattached via a substituted alkyl linker, for example an alkyl chain oflength q, where q is 1-6 carbon or hetero-atoms. The chain may be (CH₂)qwhere q is 1-6. The group may be (CH₂)₄COOH.

Alternatively, Re₁ can contain a carboxy group. The carboxy group can beattached via a substituted alkyl linker, for example an alkyl chain oflength n, where n is 1-5 carbon or hetero-atoms. The chain may be (CH₂)nwhere n is 1-5. The group may be (CH₂)₅COOH. Alternatively there may bean aryl group as part of Re₁. Re₁ may be any aryl group having a carboxydirectly attached to, or via a further alkyl chain. Re₁ may be an arylgroup substituted with CH₂COOH or CH₂COO⁻ or an amide or ester thereof.In any formula described herein, moiety

may take the form of

wherein each of Rd₁ and Rd₂ is independently H, alkyl or substitutedalkyl;each n is independently 0-6; andX is OH, O⁻ or an ester or amide.

Where the carboxy group is attached to Re₁, n can be 0. Alternativelyone of more groups can be present on the ring, for example n can be 1and Rb₁ can be halogen, sulfonamide or SO₃ ⁻. Generally compounds willnot have carboxy groups at both positions Rb1 and Re1 simultaneously asthe conjugation reactions should not be able to occur via multiple COOHmoieties.

Each Rc₁ and Rc₂ may be independently substituted with carboxy, amino,amido, sulfo or sulfonamido groups. The molecules can contain one ormore alkyl-sulfonate moieties at position Rc. Either Rc₁ and/or Rc₂ maybe alkyl-SO₃. The other Rc (Rc₁ or Rc₂) can be independently alkyl orsubstituted alkyl. Rc₁ and Rc₂ may be methyl, ethyl, propyl, butyl,pentyl, hexyl or (CH₂)_(q)SO₃H, where q is 1-6. q may be 1-4. q may be4. Rc₁ and Rc₂ may be a substituted alkyl group. Rc₁ and Rc₂ may containa COOH or —SO₃H moiety or their ester or amide derivatives.

Each of Rd₁ and Rd₂ can be independently H or methyl. Rd₁ and Rd₂ can bethe same or different. Generally Rd₁ and Rd₂ will be the same to avoidintroducing asymmetry into the molecules. Rd₁ and Rd₂ can both be H. Rd₁and Rd₂ can both be methyl.

Examples of compounds include structures according to formula (II):

or a salt thereof wherein mCat+ or mAn− is an organic or inorganicpositively/negatively charged counterion andm is an integer 0-3;each n is independently 0-6;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or a further ring fused to anadjacent carbon atom where the ring may contain SO₃ ⁻, sulfonamide,halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, COOH or an amide or ester thereofand n is 0-3; andX is OH, O⁻ or an ester or amide.

Further examples of compounds include structures according to formula(IIIa) to (IIIc):

Examples of compounds include structures according to formula (IV):

or a salt thereof wherein mCat+ or mAn− is an organic or inorganicpositively/negatively charged counterion andm is an integer 0-3;Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or a further ring fused to anadjacent carbon atom where the ring may contain SO₃ ⁻, sulfonamide,halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen, or an amide or ester thereof and nis 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl;each of Rd1 and Rd2 is independently H, alkyl or substituted alkyl;X is OH, O⁻ or an ester or amide;Y is a heteroatom selected from O, S or N; andeach of Rf₁, Rf₂, Rf₃ is independently alkyl or aryl groups.

Compounds as described herein have a Stokes shift of greater than 50 nm.The stokes shift can be greater than 100 nm, or even greater than 150.

A particularly useful compound is a nucleotide or oligonucleotidelabelled with a dye as described herein.

The labelled nucleotide or oligonucleotide may have the label via thecarboxy group to form an amide. The labelled nucleotide oroligonucleotide may have the label attached to the C5 position of apyrimidine base or the C7 position of a 7-deaza purine base through alinker moiety.

The labelled nucleotide or oligonucleotide may also have a blockinggroup covalently attached to the ribose or deoxyribose sugar of thenucleotide. The blocking group may be attached at any position on theribose or deoxyribose sugar. In particular embodiments, the blockinggroup is at the 3′ OH position of the ribose or deoxyribose sugar of thenucleotide.

Provided herein are kits including two or more nucleotides wherein atleast one nucleotide is a nucleotide labelled with a compound of thepresent disclosure. The kit may include two or more labellednucleotides. The nucleotides may be labelled with two or morefluorescent labels. Two or more of the labels may be excited using asingle excitation light source, which may be a laser or LED. Forexample, the excitation bands for the two or more labels may be at leastpartially overlapping such that excitation in the overlap region of thespectrum causes both labels to emit fluorescence. In particularembodiments, the emission from the two or more labels will occur indifferent regions of the spectrum such that presence of at least one ofthe labels can be determined by optically distinguishing the emission.

The nucleotides may be labelled with two or more fluorescent labels. Twoor more of the labels may be excited using a different excitation sourceat different wavelengths, which may be lasers. For example, the emissionbands for the two or more labels may be at least partially overlappingsuch that emission in the overlap region of the spectrum causes bothlabels to emit fluorescence at the same detection wavelength. Each labelis only excited by one of the excitation wavelengths, and thus the dyesare separately detectable due to their distinct excitation profiles. Inparticular embodiments, the excitation from the two or more labels willoccur in different regions of the spectrum such that presence of atleast one of the labels can be determined by optically distinguishingthe excitation.

Such profiling can only be carried out using labels having long stokesshifts, for example as described using the compounds as describedherein.

Once two labels can be distinguished, four separate nucleotides can beidentified using only two labels. Nucleotide 1 can be labelled withlabel 1. Nucleotide 2 can be labelled with label 2. Nucleotide 3 can belabelled with a mixture of both labels 1 and 2, and nucleotide 4 can beunlabelled (dark)

Thus the kit can include a labelled nucleotide compound as describedherein, and a further labelled nucleotide having emission at the samewavelength, but a lower Stokes shift.

Include herein is a kit comprising four nucleotides wherein a firstnucleotide is a labelled nucleotide as described herein, a secondnucleotide is labelled with a label that emits at the same wavelength asthe first labelled nucleotide, a third nucleotide is labelled with amixture of labels and the fourth is unlabelled such that each of thefour labelled nucleotides are distinguishable from each other.

The kit may contain four labelled nucleotides, where the first of fournucleotides is labelled with a compound as disclosed herein. In such akit, the second, third, and fourth nucleotides can each be labelled witha compound that is optionally different from the label on the firstnucleotide and optionally different from the labels on each other. Thus,one or more of the compounds can have a distinct absorbance maximumand/or emission maximum such that the compound(s) is(are)distinguishable from other compounds. For example, each compound canhave a distinct absorbance maximum and/or emission maximum such thateach of the compounds is distinguishable from the other three compounds.It will be understood that parts of the absorbance spectrum and/oremission spectrum other than the maxima can differ and these differencescan be exploited to distinguish the compounds. The kit may be such thattwo or more of the compounds have a distinct absorbance maximum above600 nm. The compounds of the invention typically absorb light below 500nm, but emit light at a wavelength above 600 nm

The compounds, nucleotides or kits that are set forth herein may be usedto detect, measure or identify a biological system (including, forexample, processes or components thereof). Exemplary techniques that canemploy the compounds, nucleotides or kits include sequencing, expressionanalysis, hybridisation analysis, genetic analysis, RNA analysis,cellular assay (e.g. cell binding or cell function analysis), or proteinassay (e.g. protein binding assay or protein activity assay). The usemay be on an automated instrument for carrying out a particulartechnique, such as an automated sequencing instrument. The sequencinginstrument may contain two lasers operating at different wavelengths.The sequencing instrument may have a single emission channel, which maytherefore reduce or avoid the need for multiple emission filters. Thedetection system may have a single detection channel set to a fixedemission wavelength.

Disclosed herein is a method of synthesising compounds of thedisclosure.

Prepared according to this disclosure are new starting materials, forexample (SM2), which allows synthesis of new dyes.

A compound of formula (X) or a salt thereof may be used as a startingmaterial for the synthesis of the polymethine dyes:

or a salt thereof wherein Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or afurther ring fused to an adjacent carbon atom where the ring may containSO₃ ⁻, sulfonamide, halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen or COOH and n is 0-3; each of Rc₁and Rc₂ is independently alkyl or substituted alkyl; andeach of Rd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl,or substituted aryl.

A compound of formula (X′) or a salt thereof may be used as a startingmaterial for the synthesis of the polymethine dyes:

or a salt thereof wherein x is 0-2; Ra₁ is H, SO₃ ⁻ sulfonamide,halogen, or a further ring fused to an adjacent carbon atom where thering may contain SO₃ ⁻, sulfonamide, halogen substituents;Rb₁ is SO₃ ⁻, sulphonamide, halogen or COOH and n is 0-3; each of Rc₁and Rc₂ is independently alkyl or substituted alkyl; andeach of Rd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl,or substituted aryl.

As used herein, the term “alkyl” refers to C₁-C₁₀ hydrocarbon and mayinclude C₃-C₁₀ non-aromatic carbocyclic rings. In particular embodimentsthe alkyl groups are C₁-C₆ alkyl which refers to saturated, straight- orbranched-chain hydrocarbon radicals containing between one and sixcarbon atoms, respectively. Alkyl groups may include one or moreunsaturated groups, and thus include alkenyl and alkynyl.

The term “halogen” as used herein refers to fluoro—(hereafter designatedas F), chloro—(hereafter designated as Cl), bromo—(hereafter designatedas Br) or iodo—(hereafter designated as I), and usually relates tosubstitution for a hydrogen atom in an organic compound, thissubstitution is optionally a full substitution for the hydrogen.

The term “substituted alkyl”, refers to alkyl, alkenyl or alkynyl groupsas defined above where they may optionally be further substituted with,but not limited to, halo, cyano, SO₃ ⁻, SRa, ORa, NRbRc, oxo, CONRbRc,COOH and COORb. Ra, Rb and Rc may be each independently selected from H,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl and substituted aryl. Further, saidsubstituted alkyl, substituted alkenyl and substituted alkynyl mayoptionally be interrupted by at least one hetero atom or group selectedfrom O, NRb, S(O)_(t) where t is 0 to 2, and the like. Substituted alkylalso covers group such as benzyl where the alkyl groups is comprises afurther aryl or substituted aryl moiety.

Dyes according to the present disclosure may be synthesised from avariety of different starting materials, including N-phenyl indoles.Methods for preparing polymethine dyes are well known in the art.

According to an aspect of the disclosure there are provided dyecompounds suitable for attachment to substrate moieties, particularlycomprising linker groups to enable attachment to substrate moieties.Substrate moieties can be virtually any molecule or substance to whichthe dyes of the disclosure can be conjugated and, by way of non-limitingexample, may include nucleosides, nucleotides, polynucleotides,carbohydrates, ligands, particles, solid surfaces, organic and inorganicpolymers, chromosomes, nuclei, living cells and combinations orassemblages thereof. The dyes can be conjugated by an optional linker bya variety of means including hydrophobic attraction, ionic attractionand covalent attachment. Particularly the dyes are conjugated to thesubstrate by covalent attachment. More particularly the covalentattachment is by means of a linker group.

The conjugation of the dye compound to the substrate can be carried outvia carboxyl group Rb₁ or as part of Re₁, which can be turned into anamide or ester.

The dyes according to the present disclosure may include a reactivelinker group at one of the substituent positions for covalent attachmentof the dye to another molecule. Reactive linking groups are moietiescapable of forming a bond (e.g. a covalent or non-covalent bond). In aparticular embodiment the linker may be a cleavable linker. Use of theterm “cleavable linker” is not meant to imply that the whole linker isrequired to be removed. The cleavage site can be located at a positionon the linker that results in part of the linker remaining attached tothe dye and/or substrate moiety after cleavage. Cleavable linkers maybe, by way of non-limiting example, electrophilically cleavable linkers,enzymatically cleavable linkers, nucleophilically cleavable linkers,photocleavable linkers, cleavable under reductive conditions (forexample disulfide or azide containing linkers), oxidative conditions,cleavable via use of safety-catch linkers and cleavable by eliminationmechanisms. The use of a cleavable linker to attach the dye compound toa substrate moiety provides the option of removing the label, forexample after detection, thereby avoiding any interfering signal indownstream steps.

Useful linker groups may be found in PCT publication numberWO2004/018493 (herein incorporated by reference) examples of whichinclude linkers that, may be cleaved using water-soluble phosphines orwater-soluble transition metal catalysts formed from a transition metaland at least partially water-soluble ligands. In aqueous solution thelatter form at least partially water-soluble transition metal complexes.Such cleavable linkers can be used to connect bases of nucleotides tolabels such as the dyes set forth herein.

Particular linkers may be found in PCT publication number WO2004/018493(herein incorporated by reference) such as those that include moietiesof the formula:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C1-10 substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC₁-C₁₀ substituted or unsubstituted alkyl group and * indicates wherethe moiety is connected to the remainder of the nucleotide ornucleoside).

In particular embodiments, the length of the linker between afluorescent dye (fluorophore) and a guanine base can be altered, forexample, by introducing a polyethylene glycol spacer group, therebyincreasing the fluorescence intensity compared to the same fluorophoreattached to the guanine base through other linkages known in the art.Exemplary linkers and their properties are set forth in GB patentapplication number 0517097.2, published as WO07020457, (hereinincorporated by reference). The design of linkers, and especially theirincreased length, can allow improvements in the brightness offluorophores attached to the guanine bases of guanosine nucleotides whenincorporated into polynucleotides such as DNA. Thus, when the dye is foruse in any method of analysis which employs detection of a fluorescentdye label attached to a guanine-containing nucleotide, it can beadvantageous to use a linker having a spacer group of formula—((CH₂)₂O)_(n)— wherein n is an integer between 2 and 50, for example,as described in WO07020457.

The present disclosure further provides conjugates of nucleosides andnucleotides labelled with one or more of the dyes set forth herein(modified nucleotides). Labelled nucleosides and nucleotides are usefulfor labelling polynucleotides formed by enzymatic synthesis, such as, byway of non-limiting example, in PCR amplification, isothermalamplification, solid phase amplification, polynucleotide sequencing(e.g. solid phase sequencing), nick translation reactions and the like.

Nucleosides and nucleotides may be labelled at sites on the sugar ornucleobase. As known in the art, a “nucleotide” consists of anitrogenous base, a sugar, and one or more phosphate groups. In RNA thesugar is ribose and in DNA is a deoxyribose, i.e. a sugar lacking ahydroxyl group that is present in ribose. The nitrogenous base is aderivative of purine or pyrimidine. The purines can be adenine (A) orguanine (G), and the pyrimidines can be cytosine (C), thymine (T) or inthe context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphateester of a nucleoside, with esterification occurring on the hydroxylgroup attached to the C-3 or C-5 of the sugar. Nucleotides are usuallymono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of a parent compound but which has achemical or physical modification, such as, for example, a different oradditional side group, which allows the derivative nucleotide ornucleoside to be linked to another molecule. For example, the base maybe a deazapurine. In particular embodiments, the derivatives are capableof undergoing Watson-Crick pairing. “Derivative” and “analogue” alsoinclude, for example, a synthetic nucleotide or nucleoside derivativehaving modified base moieties and/or modified sugar moieties. Suchderivatives and analogues are discussed in, for example, Scheit,Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., ChemicalReviews 90:543-584, 1990. Nucleotide analogues can also have modifiedphosphodiester linkages including phosphorothioate, phosphorodithioate,alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and thelike.

A dye may be attached to any position on a nucleotide base, for example,through a linker. In particular embodiments Watson-Crick base pairingcan still be carried out for the resulting analogue. Particularnucleobase labelling sites include the C5 position of a pyrimidine baseor the C7 position of a 7-deaza purine base. As described above a linkergroup may be used to covalently attach a dye to the nucleoside ornucleotide.

In particular embodiments the labelled nucleoside or nucleotide may beenzymatically incorporable and enzymatically extendable. Accordingly alinker moiety may be of sufficient length to connect the nucleotide tothe compound such that the compound does not significantly interferewith the overall binding and recognition of the nucleotide by a nucleicacid replication enzyme. Thus, the linker can also comprise a spacerunit. The spacer distances, for example, the nucleotide base from acleavage site or label.

Nucleosides or nucleotides labelled with dyes of the disclosure may havethe formula:

Where Dye is a dye compound according to the present disclosure, B is anucleobase, such as, for example uracil, thymine, cytosine, adenine,guanine and the like and L is an optional linker group which may or maynot be present. R′ can be H, monophosphate, diphosphate, triphosphate,thiophosphate, a phosphate ester analog, —O— attached to a reactivephosphorous containing group or —O— protected by a blocking group. R″can be H, OH, a phosphoramidite or a 3′OH blocking group and R′″ is H orOH.

Where R″ is phosphoramidite, R′ is an acid-cleavable hydroxyl protectinggroup which allows subsequent monomer coupling under automated synthesisconditions.

In a particular embodiment the blocking group is separate andindependent of the dye compound, i.e. not directly attached to it. In analternative embodiment the dye may comprise all or part of the 3′OHblocking group. Thus R can be a 3′OH blocking group which may or may notcomprise a dye compound disclosed herein.

In still yet another alternative embodiment there is no blocking groupon the 3′ carbon of the pentose sugar and the dye (or dye and linkerconstruct) attached to the base, for example, can be of a size orstructure sufficient to act as a block to the incorporation of a furthernucleotide. Thus the block can be due to steric hindrance or can be dueto a combination of size, charge and structure, whether or not the dyeis attached to the 3′ position of the sugar.

In still yet another alternative embodiment the blocking group ispresent on the 2′ or 4′ carbon of the pentose sugar and can be of a sizeor structure sufficient to act as a block to the incorporation of afurther nucleotide. The use of a blocking group allows polymerisation tobe controlled, such as by stopping extension when a modified nucleotideis incorporated. If the blocking effect is reversible, for example byway of non-limiting example by changing chemical conditions or byremoval of a chemical block, extension can be stopped at certain pointsand then allowed to continue.

In another particular embodiment a 3′OH blocking group will comprisemoieties disclosed in WO2004/018497 (herein incorporated by reference).For example the blocking group may be azidomethyl (CH₂N₃) or allyl.

In a particular embodiment a linker (between dye and nucleotide) and ablocking group are both present and are separate moieties. In particularembodiments the linker and blocking group are both cleavable undersubstantially similar conditions. Thus deprotection and deblockingprocesses may be more efficient since only a single treatment will berequired to remove both the dye compound and the block. However, in someembodiments a linker and blocking group need not be cleavable undersimilar conditions, instead being individually cleavable under distinctconditions.

This disclosure also encompasses polynucleotides incorporating dyecompounds. Such polynucleotides may be DNA or RNA comprised respectivelyof deoxyribonucleotides or ribonucleotides joined in phosphodiesterlinkage.

Polynucleotides according to the disclosure may comprise naturallyoccurring nucleotides, non-naturally occurring (or modified) nucleotidesother than the modified nucleotides of the disclosure or any combinationthereof, in combination with at least one modified nucleotide (e.g.labelled with a dye compound) set forth herein. Polynucleotidesaccording to the disclosure may also include non-natural backbonelinkages and/or non-nucleotide chemical modifications. Chimericstructures comprised of mixtures of ribonucleotides anddeoxyribonucleotides comprising at least one modified nucleotideaccording to the disclosure are also contemplated.

Modified nucleotides (or nucleosides) comprising a dye compoundaccording to the present disclosure may be used in any method ofanalysis such as methods that include detection of a fluorescent labelattached to a nucleotide or nucleoside, whether on its own orincorporated into or associated with a larger molecular structure orconjugate. In this context the term “incorporated into a polynucleotide”can mean that the 5′ phosphate is joined in phosphodiester linkage tothe 3′ hydroxyl group of a second (modified or unmodified) nucleotide,which may itself form part of a longer polynucleotide chain. The 3′ endof a modified nucleotide set forth herein may or may not be joined inphosphodiester linkage to the 5′ phosphate of a further (modified orunmodified) nucleotide. Thus, in one non-limiting embodiment thedisclosure provides a method of detecting a modified nucleotideincorporated into a polynucleotide which comprises: (a) incorporating atleast one modified nucleotide of the disclosure into a polynucleotideand (b) detecting the modified nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from the dye compoundattached to said modified nucleotide(s).

This method can include: a synthetic step (a) in which one or moremodified nucleotides according to the disclosure are incorporated into apolynucleotide and a detection step (b) in which one or more modifiednucleotide(s) incorporated into the polynucleotide are detected bydetecting or quantitatively measuring their fluorescence.

In one embodiment of the present disclosure at least one modifiednucleotide is incorporated into a polynucleotide in a synthetic step bythe action of a polymerase enzyme. However, other methods of joiningmodified nucleotides to polynucleotides, such as for example chemicaloligonucleotide synthesis or ligation of labelled oligonucleotides tounlabelled oligonucleotides can be used. Therefore, the term“incorporating”, when used in reference to a nucleotide andpolynucleotide, can encompass polynucleotide synthesis by chemicalmethods as well as enzymatic methods.

In a specific embodiment a synthetic step is carried out and mayoptionally comprise incubating a template polynucleotide strand with areaction mixture comprising fluorescently labelled modified nucleotidesof the disclosure. A polymerase can also be provided under conditionswhich permit formation of a phosphodiester linkage between a free 3′hydroxyl group on a polynucleotide strand annealed to the templatepolynucleotide strand and a 5′ phosphate group on the modifiednucleotide. Thus, a synthetic step can include formation of apolynucleotide strand as directed by complementary base-pairing ofnucleotides to a template strand.

In all embodiments of the method, the detection step may be carried outwhilst the polynucleotide strand into which the modified nucleotides areincorporated is annealed to a template strand, or after a denaturationstep in which the two strands are separated. Further steps, for examplechemical or enzymatic reaction steps or purification steps, may beincluded between a synthetic step and a detection step. In particular,the target strand incorporating the modified nucleotide(s) may beisolated or purified and then processed further or used in a subsequentanalysis. By way of example, target polynucleotides labelled withmodified nucleotide(s) in a synthetic step may be subsequently used aslabelled probes or primers. In other embodiments the product of asynthetic step set forth herein may be subject to further reaction stepsand, if desired, the product of these subsequent steps can be purifiedor isolated.

Suitable conditions for a synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodiment asynthetic step may be analogous to a standard primer extension reactionusing nucleotide precursors, including modified nucleotides set forthherein, to form an extended target strand complementary to the templatestrand in the presence of a suitable polymerase enzyme. In otherembodiments a synthetic step may itself form part of an amplificationreaction producing a labelled double stranded amplification productcomprised of annealed complementary strands derived from copying oftarget and template polynucleotide strands. Other exemplary syntheticsteps include nick translation, strand displacement polymerisation,random primed DNA labelling etc. A particularly useful polymerase enzymefor a synthetic step is one that is capable of catalysing theincorporation of one or more of the modified nucleotides set forthherein. A variety of naturally occurring or modified polymerases can beused. By way of example, a thermostable polymerase can be used for asynthetic reaction that is carried out using thermocycling conditions,whereas a thermostable polymerase may not be desired for isothermalprimer extension reactions. Suitable thermostable polymerases which arecapable of incorporating the modified nucleotides according to thedisclosure include those described in WO 2005/024010 or WO06120433, eachof which is incorporated herein by reference. In synthetic reactionswhich are carried out at lower temperatures such as 37° C., polymeraseenzymes need not necessarily be thermostable polymerases, therefore thechoice of polymerase will depend on a number of factors such as reactiontemperature, pH, strand-displacing activity and the like.

In specific non-limiting embodiments the disclosure encompasses methodsof nucleic acid sequencing, re-sequencing, whole genome sequencing,single nucleotide polymorphism scoring, or any other applicationinvolving the detection of the modified nucleotide or nucleosidelabelled with dyes set forth herein when incorporated into apolynucleotide. Any of a variety of other applications benefitting fromthe use of polynucleotides labelled with the modified nucleotidescomprising fluorescent dyes can use modified nucleotides or nucleosideslabelled with dyes set forth herein.

In a particular embodiment the disclosure provides use of modifiednucleotides comprising dye compounds according to the disclosure in apolynucleotide sequencing-by-synthesis reaction. Sequencing-by-synthesisgenerally involves sequential addition of one or more nucleotides oroligonucleotides to a growing polynucleotide chain in the 5′ to 3′direction using a polymerase or ligase in order to form an extendedpolynucleotide chain complementary to the template nucleic acid to besequenced. The identity of the base present in one or more of the addednucleotide(s) can be determined in a detection or “imaging” step. Theidentity of the added base may be determined after each nucleotideincorporation step. The sequence of the template may then be inferredusing conventional Watson-Crick base-pairing rules. The use of themodified nucleotides labelled with dyes set forth herein fordetermination of the identity of a single base may be useful, forexample, in the scoring of single nucleotide polymorphisms, and suchsingle base extension reactions are within the scope of this disclosure.

In an embodiment of the present disclosure, the sequence of a templatepolynucleotide is determined by detecting the incorporation of one ormore nucleotides into a nascent strand complementary to the templatepolynucleotide to be sequenced through the detection of fluorescentlabel(s) attached to the incorporated nucleotide(s). Sequencing of thetemplate polynucleotide can be primed with a suitable primer (orprepared as a hairpin construct which will contain the primer as part ofthe hairpin), and the nascent chain is extended in a stepwise manner byaddition of nucleotides to the 3′ end of the primer in apolymerase-catalysed reaction.

In particular embodiments each of the different nucleotide triphosphates(A, T, G and C) may be labelled with a unique fluorophore and alsocomprises a blocking group at the 3′ position to prevent uncontrolledpolymerisation. Alternatively one of the four nucleotides may beunlabelled (dark). The polymerase enzyme incorporates a nucleotide intothe nascent chain complementary to the template polynucleotide, and theblocking group prevents further incorporation of nucleotides. Anyunincorporated nucleotides can be washed away and the fluorescent signalfrom each incorporated nucleotide can be “read” optically by suitablemeans, such as a charge-coupled device using laser excitation andsuitable emission filters. The 3′-blocking group and fluorescent dyecompounds can then be removed (deprotected), (simultaneously orsequentially) to expose the nascent chain for further nucleotideincorporation. Typically the identity of the incorporated nucleotidewill be determined after each incorporation step but this is notstrictly essential. Similarly, U.S. Pat. No. 5,302,509 (which isincorporated herein by reference) discloses a method to sequencepolynucleotides immobilised on a solid support.

The method, as exemplified above, utilizes the incorporation offluorescently labelled, 3′-blocked nucleotides A, G, C and T into agrowing strand complementary to the immobilised polynucleotide, in thepresence of DNA polymerase. The polymerase incorporates a basecomplementary to the target polynucleotide, but is prevented fromfurther addition by the 3′-blocking group. The label of the incorporatednucleotide can then be determined and the blocking group removed bychemical cleavage to allow further polymerisation to occur. The nucleicacid template to be sequenced in a sequencing-by-synthesis reaction maybe any polynucleotide that it is desired to sequence. The nucleic acidtemplate for a sequencing reaction will typically comprise a doublestranded region having a free 3′ hydroxyl group which serves as a primeror initiation point for the addition of further nucleotides in thesequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′ OH group for initiation of the sequencing reaction. Insuch embodiments sequencing may proceed by strand displacement. Incertain embodiments a primer bearing the free 3′ hydroxyl group may beadded as a separate component (e.g. a short oligonucleotide) whichhybridises to a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intra-molecular duplex, such as for example ahairpin loop structure. Hairpin polynucleotides and methods by whichthey may be attached to solid supports are disclosed in Internationalapplication publication nos. WO0157248 and WO2005/047301, each of whichis incorporated herein by reference. Nucleotides can be addedsuccessively to a growing primer, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of the basewhich has been added may be determined, particularly but not necessarilyafter each nucleotide addition, thus providing sequence information forthe nucleic acid template. Thus, a nucleotide is incorporated into anucleic acid strand (or polynucleotide) by joining of the nucleotide tothe free 3′ hydroxyl group of the nucleic acid strand via formation of aphosphodiester linkage with the 5′ phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart, for example via covalent attachment. In certain embodimentstemplate polynucleotides may be attached directly to a solid support(e.g. a silica-based support). However, in other embodiments of thedisclosure the surface of the solid support may be modified in some wayso as to allow either direct covalent attachment of templatepolynucleotides, or to immobilise the template polynucleotides through ahydrogel or polyelectrolyte multilayer, which may itself benon-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO00006770(incorporated herein by reference), wherein polynucleotides areimmobilised on a glass support by reaction between a pendant epoxidegroup on the glass with an internal amino group on the polynucleotide.In addition, polynucleotides can be attached to a solid support byreaction of a sulphur-based nucleophile with the solid support, forexample, as described in WO2005/047301 (incorporated herein byreference). A still further example of solid-supported templatepolynucleotides is where the template polynucleotides are attached tohydrogel supported upon silica-based or other solid supports, forexample, as described in WO00/31148, WO01/01143, WO02/12566,WO03/014392, U.S. Pat. No. 6,465,178 and WO00/53812, each of which isincorporated herein by reference.

A particular surface to which template polynucleotides may beimmobilised is a polyacrylamide hydrogel. Polyacrylamide hydrogels aredescribed in the references cited above and in WO2005/065814, which isincorporated herein by reference.

DNA template molecules can be attached to beads or microparticles, forexample as described in U.S. Pat. No. 6,172,218 (which is incorporatedherein by reference). Attachment to beads or microparticles can beuseful for sequencing applications. Bead libraries can be prepared whereeach bead contains different DNA sequences. Exemplary libraries andmethods for their creation are described in Nature. 437, 376-380 (2005);Science. 309, 5741, 1728-1732 (2005), each of which is incorporatedherein by reference. Sequencing of arrays of such beads usingnucleotides set forth herein is within the scope of the disclosure.

Template(s) that are to be sequenced may form part of an “array” on asolid support, in which case the array may take any convenient form.Thus, the method of the disclosure is applicable to all types of highdensity arrays, including single-molecule arrays, clustered arrays andbead arrays. Modified nucleotides labelled with dye compounds of thepresent disclosure may be used for sequencing templates on essentiallyany type of array, including but not limited to those formed byimmobilisation of nucleic acid molecules on a solid support.

However, the modified nucleotides labelled with dye compounds of thedisclosure are particularly advantageous in the context of sequencing ofclustered arrays. In clustered arrays, distinct regions on the array(often referred to as sites, or features) comprise multiplepolynucleotide template molecules. Generally, the multiplepolynucleotide molecules are not individually resolvable by opticalmeans and are instead detected as an ensemble. Depending on how thearray is formed, each site on the array may comprise multiple copies ofone individual polynucleotide molecule (e.g. the site is homogenous fora particular single- or double-stranded nucleic acid species) or evenmultiple copies of a small number of different polynucleotide molecules(e.g. multiple copies of two different nucleic acid species). Clusteredarrays of nucleic acid molecules may be produced using techniquesgenerally known in the art. By way of example, WO 98/44151 andWO00/18957, each of which is incorporated herein, describe methods ofamplification of nucleic acids wherein both the template andamplification products remain immobilised on a solid support in order toform arrays comprised of clusters or “colonies” of immobilised nucleicacid molecules. The nucleic acid molecules present on the clusteredarrays prepared according to these methods are suitable templates forsequencing using the modified nucleotides labelled with dye compounds ofthe disclosure.

The modified nucleotides labelled with dye compounds of the presentdisclosure are also useful in sequencing of templates on single moleculearrays. The term “single molecule array” or “SMA” as used herein refersto a population of polynucleotide molecules, distributed (or arrayed)over a solid support, wherein the spacing of any individualpolynucleotide from all others of the population is such that it ispossible to individually resolve the individual polynucleotidemolecules. The target nucleic acid molecules immobilised onto thesurface of the solid support can thus be capable of being resolved byoptical means in some embodiments. This means that one or more distinctsignals, each representing one polynucleotide, will occur within theresolvable area of the particular imaging device used.

Single molecule detection may be achieved wherein the spacing betweenadjacent polynucleotide molecules on an array is at least 100 nm, moreparticularly at least 250 nm, still more particularly at least 300 nm,even more particularly at least 350 nm. Thus, each molecule isindividually resolvable and detectable as a single molecule fluorescentpoint, and fluorescence from said single molecule fluorescent point alsoexhibits single step photobleaching.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualised, it is possible to distinguishone molecule on the array from its neighbouring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to published applications WO00/06770 and WO 01/57248, each ofwhich is incorporated herein by reference. Although one use of themodified nucleotides of the disclosure is in sequencing-by-synthesisreactions, the utility of the modified nucleotides is not limited tosuch methods. In fact, the nucleotides may be used advantageously in anysequencing methodology which requires detection of fluorescent labelsattached to nucleotides incorporated into a polynucleotide.

In particular, the modified nucleotides labelled with dye compounds ofthe disclosure may be used in automated fluorescent sequencingprotocols, particularly fluorescent dye-terminator cycle sequencingbased on the chain termination sequencing method of Sanger andco-workers. Such methods generally use enzymes and cycle sequencing toincorporate fluorescently labelled dideoxynucleotides in a primerextension sequencing reaction. So called Sanger sequencing methods, andrelated protocols (Sanger-type), utilize randomised chain terminationwith labelled dideoxynucleotides.

Thus, the present disclosure also encompasses modified nucleotideslabelled with dye compounds which are dideoxynucleotides lackinghydroxyl groups at both of the 3′ and 2′ positions, such modifieddideoxynucleotides being suitable for use in Sanger type sequencingmethods and the like.

Modified nucleotides labelled with dye compounds of the presentdisclosure incorporating 3′ blocking groups, it will be recognized, mayalso be of utility in Sanger methods and related protocols since thesame effect achieved by using modified dideoxy nucleotides may beachieved by using modified nucleotides having 3′-OH blocking groups:both prevent incorporation of subsequent nucleotides. Where nucleotidesaccording to the present disclosure, and having a 3′ blocking group areto be used in Sanger-type sequencing methods it will be appreciated thatthe dye compounds or detectable labels attached to the nucleotides neednot be connected via cleavable linkers, since in each instance where alabelled nucleotide of the disclosure is incorporated; no nucleotidesneed to be subsequently incorporated and thus the label need not beremoved from the nucleotide.

The present disclosure also provides kits including modified nucleosidesand/or nucleotides labelled with dyes. Such kits will generally includeat least one modified nucleotide or nucleoside labelled with a dye setforth herein together with at least one further component. The furthercomponent(s) may be one or more of the components identified in a methodset forth above or in the Examples section below. Some non-limitingexamples of components that can be combined into a kit of the presentdisclosure are set forth below.

In a particular embodiment, a kit can include at least one modifiednucleotide or nucleoside labelled with a dye set forth herein togetherwith modified or unmodified nucleotides or nucleosides. For example,modified nucleotides labelled with dyes according to the disclosure maybe supplied in combination with unlabelled or native nucleotides, and/orwith fluorescently labelled nucleotides or any combination thereof.Accordingly the kits may comprise modified nucleotides labelled withdyes according to the disclosure and modified nucleotides labelled withother, for example, prior art dye compounds. Combinations of nucleotidesmay be provided as separate individual components (e.g. one nucleotidetype per vessel or tube) or as nucleotide mixtures (e.g. two or morenucleotides mixed in the same vessel or tube).

Where kits comprise a plurality, particularly two, more particularlyfour, modified nucleotides labelled with a dye compound, the differentnucleotides may be labelled with different dye compounds, or one may bedark, with no dye compounds, or one may be a mixture of two dyecompounds. Where the different nucleotides are labelled with differentdye compounds it is a feature of the kits that said dye compounds arespectrally distinguishable fluorescent dyes. As used herein, the term“spectrally distinguishable fluorescent dyes” refers to fluorescent dyesthat emit fluorescent energy at wavelengths that can be distinguished byfluorescent detection equipment (for example, a commercial capillarybased DNA sequencing platform) when two or more such dyes are present inone sample. When two modified nucleotides labelled with fluorescent dyecompounds are supplied in kit form, it is a feature of some embodimentsthat the spectrally distinguishable fluorescent dyes can be excited atthe same wavelength, such as, for example by the same laser.Alternatively, it is a feature of some embodiments that the spectrallydistinguishable fluorescent dyes can be excited at differentwavelengths, such as, for example by different lasers, but emit at thesame wavelength. When four modified nucleotides labelled withfluorescent dye compounds are supplied in kit form, it is a feature ofsome embodiments that two of the spectrally distinguishable fluorescentdyes can both be excited at one wavelength and the other two spectrallydistinguishable dyes can both be excited at another wavelength.Particular excitation wavelengths are 532 nm, 630 nm to 700 nm,particularly 660 nm.

In one embodiment a kit includes a modified nucleotide labelled with acompound of the present disclosure and a second modified nucleotidelabelled with a second dye wherein the dyes have a difference inabsorbance maximum of at least 100 nm. More particularly one of the dyecompounds has a Stokes shifts of between 15-40 nm and a compound of theinvention has a stokes shift of at least 50 nm, or greater than 100 nm.Compounds of the invention can have Stokes shifts of greater than 50 nm,greater than 100 nm or even greater than 150 nm.

In a further embodiment a kit can further include modified nucleotideslabelled with fluorescent dyes wherein the dyes are excited at saygreater than 600 nm nm. The dyes can have a difference in absorbancemaximum of at least 100 nm. Still yet more particularly the second dyecompound can have a different absorbance maximum above 600 nm,particularly above 630 nm. Particular dyes which are spectrallydistinguishable from polymethine dyes of the present disclosure andwhich meet the above criteria are polymethine analogues as described inU.S. Pat. No. 5,268,486 (for example Cy5) or WO 0226891 (Alexa 647;Molecular Probes/Life technologies A20006) or unsymmetrical polymethinesas disclosed in U.S. Pat. No. 6,924,372, each of which is incorporatedherein by reference.

In an alternative embodiment, the kits of the disclosure may containnucleotides where the same base is labelled with two differentcompounds. A first nucleotide may be labelled with a compound of thedisclosure. A second nucleotide may be labelled with a spectrallydistinct compound, for example a ‘red’ dye absorbing at greater than 600nm. A third nucleotide may be labelled as a mixture of the compound ofthe disclosure and the spectrally distinct compound, and the fourthnucleotide may be ‘dark’ and contain no label. In simple terms thereforethe nucleotides 1-4 may be labelled ‘green’, ‘red’, ‘red/green’, anddark. To simplify the instrumentation further, four nucleotides can belabelled with a two dyes excited with a single laser, and thus thelabelling of nucleotides 1-4 may be ‘red 1’, ‘red 2’ ‘red 1/red 2’, anddark.

Nucleotides may contain two dyes of the present disclosure. Dyes whereRa₁ is a further aromatic ring fused to adjacent carbons of the indolering absorb at a longer wavelength than where the dyes do not have thefurther aromatic conjugation. A kit may contain two or more nucleotideslabelled with dyes of the disclosure. A kit may contain a nucleotidelabelled with a compound of the disclosure where Ra₁ is H, SO₃ ⁻,sulphonamide or halogen, and one nucleotide labelled with a compound ofthe disclosure where Ra₁ is a further ring fused to an adjacent carbonatom. Kits may contain a further nucleotide where the nucleotide islabelled with a dye that absorbs in the region of 520 nm to 560 nm. Kitsmay further contain an unlabelled nucleotide.

Although kits are exemplified above in regard to configurations havingdifferent nucleotides that are labelled with different dye compounds, itwill be understood that kits can include 2, 3, 4 or more differentnucleotides that have the same dye compound.

In particular embodiments a kit may include a polymerase enzyme capableof catalyzing incorporation of the modified nucleotides into apolynucleotide. Other components to be included in such kits may includebuffers and the like. The modified nucleotides labelled with dyesaccording to the disclosure, and other any nucleotide componentsincluding mixtures of different nucleotides, may be provided in the kitin a concentrated form to be diluted prior to use. In such embodiments asuitable dilution buffer may also be included. Again, one or more of thecomponents identified in a method set forth herein can be included in akit of the present disclosure.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessexpressly and unequivocally limited to one referent. It will be apparentto those skilled in the art that various modifications and variationscan be made to various embodiments described herein without departingfrom the spirit or scope of the present teachings. Thus, it is intendedthat the various embodiments described herein cover other modificationsand variations within the scope of the appended claims and theirequivalents.

DESCRIPTION OF FIGURES

FIG. 1 illustrates fluorescence intensities of nucleotide labelled witha new dye (NR5201s), an example of the type disclosed herein, and astructural analogue (NR550S0) when their solutions excited at 540 nm.NR550S0 (structure shown below) is a polymethine dye having an indole atboth ends of the polymethine chain, as is typical of fluorescent dyeshaving a stokes shift in the region of 40-50 nm. The dye having thestokes shift of 40-50 nm shows a higher fluorescence signal than thelong stokes shift dye.

FIG. 2 illustrates fluorescence intensities of FFN's based on new dye(NR5201s) and its commercial structural analogue (NR550S0) when theirsolutions excited at 460 nm. Unlike at 540 nm, the long stokes shift dyeis brighter when excited at 460 nm. Thus the two labels can bedifferentiated when measured at 590 nm emission based in the excitationwavelength.

EXPERIMENTAL DETAILS SM2

These starting materials were prepared from pyrilium salts (SM1) usingethyl orthoformmate or its derivatives

Example 1 SM2-1

2,4,6-Trimethylpyrilium tetrafluoroborate (1 ekv), triethylortoformate(1.5 ekv) and N,N′-diphenylformamidine (1.1. ekv) in acetic acid wereheated for 6 h at 80° C. Reaction mixture was left overnight at roomtemperature and the product was filtered off as yellow crystals. Yield87%.

Indopyrilocyanines (X).

These starting materials were prepared from pyrilium salts derivatives(SM2) using N-substituted indolium salts

Example 2 (X)-1

Equimolar amounts of SM2-1 and N-Phenyl-2,3,3-trimethylindolium salt inmixture of acetic acid, acetic anhydride and pyridine (1:1:0.5) werestirred for 3 h at 80° C.

The product was precipitated with diethyl ether and column purified.Yield 48%.

Indopyridocyanines

These dyes were prepared from pyrilium derivatives (X) using substitutedamines or their salts

Example 3-1 (NR5201s)

Starting materials were stirred for 5 min in ethanol thenN-ethyl-N,N-diisopropylamine added and stirring was continued for 0.5 h.After solvent evaporation the dye was collected, washed with water.Yield 95%.

Example 3-2

Starting materials were stirred for 5 min in ethanol thenN-ethyl-N,N-diisopropylamine added. Stirring was continued for 0.5 h.After solvent evaporation the dye was collected, washed with water.Yield ˜95%.

Example 3-3

Starting materials were mixed in ethanol thenN-ethyl-N,N-diisopropylamine added. Reaction mixture was stirred for 0.5h. After solvent evaporation the dye was collected, washed with water.Yield ˜95%.

Synthesis of Dye Labelled Nucleotide Triphosphates

Dye Conjugate pppT-NR5201s

Preparation:

Anhydrous DMa (5 mL) and Hunig's Base (0.06 mL) were added to the driedsample of the dye (3-1) (80 mg). A solution of TSTU, (0.25 g) in 5 mL ofdry DMA was then added to this. The reaction mixture was stirred at roomtemperature for 1 h. After activation was completed (TLC: 15% H₂O inCH₃CN) this solution was added to the solution of pppT-LN3 (0.23 g) inwater (7 mL). The reaction mixture was stirred at room temperature undernitrogen atmosphere for 3 h. The reaction mixture was cooled down to ˜4°C. with an ice-bath, then a solution of 0.1 M TEAB (5 mL) in water wasadded and the mixture was stirred at room temperature for 10 min. Thereaction mixture was applied to column with ˜75 g of DEAE Sephadex resinsuspension in 0.05 M TEAB solution in water and washed with TEAB(concentration gradient from 0.10 M up to 0.75 M). Red colouredfractions were collected, the solvent evaporated and then the residueco-evaporated again with water to remove more TEAB and vac down todryness. The dye was then re-dissolved in TEAB 0.1 M. This solution wasfiltered through a syringe filter 0.2 nm pore size and the product waspurified by HPLC using C18 reverse phase column with acetonitrile-0.1 MTEAB. Yield 78%.

Dye Conjugate pppA-NR5201s

Preparation:

Anhydrous DMA (5 mL) and Hunig's Base (0.06 mL) were added to the driedsample of the dye (3-1) (80 mg). A solution of TSTU, (0.25 g) in 5 mL ofdry DMA was then added to this. The reaction mixture was stirred at roomtemperature for 2 h. After activation was completed (TLC: 15% H₂O inCH₃CN) this solution was added to the solution of pppA-LN3 (0.25 g) inwater (7 mL). The reaction mixture was stirred at room temperature undernitrogen atmosphere for 24 h. The reaction mixture was cooled down to˜4° C. with an ice-bath, then a solution of 0.1 M TEAB (5 mL) in waterwas added and the mixture was stirred at room temperature for 10 min.The reaction mixture was applied to column with ˜75 g of DEAE Sephadexresin suspension in 0.05 M TEAB solution in water and washed with TEAB(concentration gradient from 0.10 M up to 0.75 M). Red colouredfractions were collected, the solvent evaporated and then the residueco-evaporated again with water to remove more TEAB and vac down todryness. The dye was then re-dissolved in TEAB 0.1 M. This solution wasfiltered through a syringe filter 0.2 nm pore size and the product waspurified by HPLC using C18 reverse phase column with acetonitrile-0.1 MTEAB. Yield 75%.

Fluorescence Properties

FIG. 1 illustrates fluorescence intensities of nucleotide labelled witha new dye (NR5201s), an example of the type disclosed herein, and astructural analogue (NR550S0) when their solutions excited at 540 nm.NR550S0 (structure shown below) is a polymethine dye having an indole atboth ends of the polymethine chain, as is typical of fluorescent dyeshaving a stokes shift in the region of 40-50 nm. The dye having thestokes shift of 40-50 nm shows a higher fluorescence signal than thelong stokes shift dye.

FIG. 2 illustrates fluorescence intensities of FFN's based on new dye(NR5201s) and its commercial structural analogue (NR550S0) when theirsolutions excited at 460 nm. Unlike at 540 nm, the long stokes shift dyeis brighter when excited at 460 nm. Thus the two labels can bedifferentiated when measured at 590 nm emission based in the excitationwavelength.

From the comparison of ratios of fluorescence intensities of a new dyeNR5201s with long Stokes shift and its structural analogue with a normalstokes shift, one can see the advantage of using new dyes due to avariance in signal intensities when solutions are excited at differentwavelengths.

In the charts above, for dye NR550S0 the ratio of fluorescenceintensities at 540 nm (193.0) and at 460 nm (10.1) is 19.3 as the dyedoes not absorb efficiently at 460 nm. In the same conditions for thenew dye NR5201s, the ratio of fluorescence intensities at 540 nm (93.0)and 460 nm (40.1) is only about 2 as the longer stokes shift means thedye has a much higher level or absorbance at 460 nm. Due to these uniqueproperties, the new dyes as disclosed herein allow more efficient dataanalysis as the signal to noise is improved, and allow sequencingplatforms to operate using fewer than the conventional four detectionchannels.

1. A compound of formula (I′) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and m is an integer 0-3; x is an integer 0-2; Ra₁ isH, SO₃ ⁻, sulfonamide, halogen, hydroxy, alkoxy, amino or a further ringfused to an adjacent carbon atom where the ring may contain SO₃ ⁻,sulfonamide, halogen substituents; Rb₁ is SO₃ ⁻, sulphonamide, halogen,hydroxy, alkoxy, amino, COOH or an amide or ester thereof and n is 0-3;each of Rc₁ and Rc₂ is independently alkyl or substituted alkyl; each ofRd₁ and Rd₂ is independently H, alkyl, aryl, substituted alkyl, orsubstituted aryl; and Re₁ is alkyl, substituted alkyl, aryl orsubstituted aryl; wherein either Rc₁, Rb₁ or Re₁ contains a COOH or COO⁻or an amide or ester thereof.
 2. A compound according to claim 1 offormula (I) or mesomeric forms thereof:

wherein mCat+ or mAn− is an organic or inorganic positively/negativelycharged counterion and m is an integer 0-3; Ra₁ is H, SO₃ ⁻,sulfonamide, halogen, hydroxy, alkoxy, amino or a further ring fused toan adjacent carbon atom where the ring may contain SO₃ ⁻, sulfonamide,halogen substituents; Rb₁ is SO₃ ⁻, sulphonamide, halogen, hydroxy,alkoxy, amino, COOH or an amide or ester thereof and n is 0-3; each ofRc₁ and Rc₂ is independently alkyl or substituted alkyl; each of Rd₁ andRd₂ is independently H, alkyl, aryl, substituted alkyl, or substitutedaryl; and Re₁ is alkyl, substituted alkyl, aryl or substituted aryl;wherein either Rc₁, Rb₁ or Re₁ contains a COOH or COO⁻ or an amide orester thereof.
 3. A compound according to claim 1 or claim 2 wherein Re₁is alkyl and Rb₁ is COOH or COO⁻ or an amide or ester thereof.
 4. Acompound according to claim 1 or claim 2 wherein Re₁ is an alkyl groupsubstituted with COOH or COO⁻ or an amide or ester thereof.
 5. Acompound according to claim 1 or claim 2 wherein Re₁ is an aryl groupsubstituted with COOH or COO⁻ or an amide or ester thereof.
 6. Acompound according to claim 1 or claim 2 wherein Re₁ is an aryl groupsubstituted with COOH or COO⁻ or an amide or ester thereof wherein thereis an alkyl group between the aryl ring and the COOH or COO⁻ or an amideor ester thereof.
 7. A compound according to claim 1 or claim 2 whereinRe₁ is an aryl group substituted with CH₂COOH or CH₂COO⁻ or an amide orester thereof.
 8. A compound according to any one of claims 4 to 7wherein n is
 0. 9. A compound according to any one of claims 4 to 7wherein n is 1 and Rb₁ is SO₃ ⁻ or sulphonamide.
 10. A compoundaccording to any one of claims 1 to 9 wherein each of Rc₁ and Rc₂ ismethyl.
 11. A compound according to any one of claims 1 to 9 wherein Rc₁or Rc₂ is methyl, ethyl, propyl or —(CH₂)_(q)SO₃ ⁻ where q is 1-6.
 12. Acompound according to claim 11 wherein either Rc₁ or Rc₂ is (CH₂)₄—SO₃⁻.
 13. A compound according to any one preceding claim wherein Ra₁ is H,SO₂NH₂ or SO₃ ⁻.
 14. A compound according to any one of claims 1 to 12wherein Ra₁ is a ring fused to an adjacent carbon atom.
 15. A compoundaccording to claim 14 wherein the ring contains one or more SO₃ ⁻ orsulphonamide substituents.
 16. A compound according to any one precedingclaim wherein each of Rd₁ and Rd₂ is independently H or methyl.
 17. Acompound according to claim 1 which is represented by formula (II):

or a salt thereof wherein mCat+ or mAn− is an organic or inorganicpositively/negatively charged counterion and m is an integer 0-3; each nis independently 0-6; Ra₁ is H, SO₃ ⁻, sulfonamide, halogen, or afurther ring fused to an adjacent carbon atom where the ring may containSO₃ ⁻, sulfonamide, halogen substituents; Rb₁ is SO₃ ⁻, sulphonamide,halogen, COOH or an amide or ester thereof and n is 0-3; and X is OH, O⁻or an ester or amide.
 18. A compound according to any one precedingclaim wherein the compound is attached to a nucleotide oroligonucleotide.
 19. A nucleotide or oligonucleotide labelled with acompound according to claims 1 to
 17. 20. A labelled nucleotide oroligonucleotide according to claim 18 wherein the label is attached viaan amide linkage formed from the COOH moiety.
 21. A labelled nucleotideor oligonucleotide according to claims 18 or 19 wherein the label isattached to the C5 position of a pyrimidine base or the C7 position of a7-deaza purine base through a linker moiety.
 22. A labelled nucleotideor oligonucleotide according to claims 19 to 21, further comprising a 3′OH blocking group covalently attached to the ribose or deoxyribose sugarof the nucleotide.
 23. A kit comprising two or more nucleotides whereinat least one nucleotide is a labelled nucleotide according to claims 19to
 22. 24. A kit according to claim 22 wherein two of the labellednucleotides are measured by detection at the same wavelength.
 25. A kitaccording to claim 24 comprising four nucleotides wherein a firstnucleotide is a labelled nucleotide according to claims 18 to 21, asecond nucleotide is labelled with a label that emits at the samewavelength as the first labelled nucleotide, a third nucleotide islabelled with a mixture of labels and the fourth is unlabelled such thateach of the four labelled nucleotides are distinguishable from eachother.
 26. A kit according to claim 22 to 25 wherein the first labellednucleotide has a Stokes shift of greater than 100 nm and the secondlabelled nucleotide has a Stokes shift of less than 50 nm.
 27. Use of anucleotide according to any one of claims 19 to 22, an oligonucleotideaccording to claims 19 to 22 or a kit according to any one of claims 23to 26 in sequencing, expression analysis, hybridisation analysis,genetic analysis, RNA analysis or protein binding assays.
 28. Useaccording to claim 27 on an automated sequencing instrument wherein saidautomated sequencing instrument comprises two lasers operating atdifferent wavelengths and a detection system having a single detectionchannel set to a fixed emission wavelength.
 29. A method of synthesisinga compound according to claims 1 to 17 utilising the following startingmaterial:

or a salt thereof wherein x is 0-2; Ra₁ is H, SO₃ ⁻, sulfonamide,halogen, or a further ring fused to an adjacent carbon atom where thering may contain SO₃ ⁻, sulfonamide, halogen substituents; Rb₁ is SO₃ ⁻,sulphonamide, halogen or COOH and n is 0-3; each of Rc₁ and Rc₂ isindependently alkyl or substituted alkyl; and each of Rd₁ and Rd₂ isindependently H, alkyl, aryl, substituted alkyl, or substituted aryl.